Heat shock proteins (HSPs) are known conventionally as stress proteins or protein chaperones which play fundamental housekeeping roles in all cellular events involving protein folding and unfolding (Morimoto R. I., 1998 Genes Dev 12:3788-3796). HSPs are multifunctional proteins and facilitate various processes including vesicular transport, signaling, and so forth. The HSP gp96 (which stands for glycoprotein of 96 kDa) also known as grp94, endoplasmin or ERp99, belongs to the HSP90 family of heat shock proteins. It is constitutively expressed and normally resides in the lumen of the endoplasmic reticulum (ER), where its chaperone function is vital for the proper folding of many substrates. In humans, only one gene locus has been mapped on chromosome 12 and was named tra-1. It encodes a protein of 803 amino acids, which contains an ER-retention sequence, KDEL, at its C-terminus. Like other HSPs, gp96 is induced by the accumulation of misfolded proteins, it binds and hydrolyzes ATP and chaperones multiple protein substrates. The crucial role of gp96 as a housekeeping gene is emphasized by the fact that gp96-gene knockout mice are embryonic lethal (Li Z. et al., 2002 Frontiers in Bioscience 7:731-751).
Gp96 plays a critical role in presentation of exogenous antigens by MHC class I, by acting as a chaperone to such antigens, which then enter the cells through endocytosis. Subsequently, the antigenic peptide is transported into the endoplasmic reticulum where it is charged onto a cognate MHC class I molecule. That process, which is referred to as cross-presentation, leads to the presentation of MHC I-peptide complexes on the cell surface leading to stimulation of CD8+ T cells (Suto R. & P. K. Srivastava 1995 Science 269:1585-1588). Thus, gp96 purified from cells chaperones antigenic peptides generated in that cell. Immunization with gp96-peptide complexes purified from tumors or pathogen-infected cells elicits specific immunity directed against the tumor or pathogen, respectively (Janetzki, S. et al., 1998 J. Immunother. 21:269-276).
Gp96 is the most intensely studied HSP from the immunological point of view, and has been shown to carry out many functions such as activating both innate and adaptive immunity, peptide antigen presentation, transfer of such peptides to MHC molecules, activation of antigen-presenting cells (APCs), and playing an important role in tumor immunity. gp96 also has several peptide-independent activities, including a critical role in the assembly of functional Toll-like receptors (TLRs), and acting as a danger signal by activating dendritic cells (DCs) to secrete proinflammatory cytokines and chemokines (Li Z. et al., 2002 Curr Opin Immunol 14:45-51; Srivastava, P. 2002 Nat Rev Immunol 2:185-194; Hilf N. et al 2002 Int. J. Hyperthermia 18:521-533). These combined features and multifunctional characteristics make gp96 a powerful weapon, and accordingly it has also been labeled “the Swiss Army Knife of the Immune System” (Schild and Rammensee 2000 Nat. Immunol. 1:100-101).
HSPs perform diverse functions in two alternative modes of inflammation: sterile inflammation, which results from endogenous stimuli and is necessary for body maintenance, and septic inflammation, which protects us from environmental pathogens. Endogenous HSPs, such as gp96, are key players in the modulation of these two modes of inflammation, and as such, they are potential targets for novel therapies for cancer, infections and autoimmunity (Quintana and Cohen, 2005 J. Immunol. 175: 2777-2782)
Recent studies provide new insights into the role of gp96 in the assembly of functional Toll-like receptors (TLRs). TLRs are an important family of receptors that contribute to innate immunity and regulate adaptive immunity. These pattern-recognition receptors are able to recognize unique structural entities such as bacterial lipoproteins (TLR2 in heterodimers with TLR1 or TLR6), double-stranded RNA (TLR3), lipopolysaccharide (LPS) (TLR4), bacterial flagellin (TLR5), certain pathogen-associated RNA sequences (TLR7), and pathogen-associated unmethylated CpG motifs in DNA (TLR9) (Iwasaki, A. & Medzhitov, R. 2004 Nat Immunol. 5:987-995). Defects in TLR expression or function can lead to increased susceptibility to infection with various pathogens. In contrast, excessive or inappropriate TLR signaling is associated with pathological processes, like LPS-induced endotoxin shock in sepsis, certain autoimmune and inflammatory conditions and cancer. Thus, mechanisms that regulate TLR expression and function may be critical for shaping both immunity to pathogens and pathologic immune reactions.
New evidence reveals that gp96 is the unique and obligatory master chaperone for TLRs. Intact gp96 is essential for signaling by TLR1, TLR2, TLR3, TLR4, TLR5, TLR7 and TLR9. In the absence of gp96, TLRs are not functional, are largely retained in the endoplasmic reticulum, and cannot mediate responses such as TLR4-induced endotoxin shock or induction of cytokines and host resistance by Listeria monocytogenes (Yang Y., et al. 2007 Immunity 26:215-226). In addition to its role as a chaperone for TLRs, gp96 has a role in the amplification of dendritic cell activation by bacterial products (Warger T. et al 2006 J. Biol. Chem. 281:22545-22553).
Enforcing cell surface expression of gp96 in a transgenic mouse model induced significant activation of dendritic cells and spontaneous lupus-like autoimmune disease. The development of such autoimmunity is dependent on MyD88, an important downstream adaptor protein for signaling by TLRs (Liu B. et al., 2003 Proc Natl Acad Sci USA 100:15824-15829). Similarly, disturbing ER retention of gp96 showed dendritic cell activation attributable to increased gp96 surface presentation and lupus-like autoimmune phenotypes (Han J. M. et al, 2007 Am. J. Pathol. 170:2042-2054). Hence, chronic activation of dendritic cells by gp96 may cause breakdown of peripheral tolerance, resulting in autoimmune disease. Indistinguishable results were obtained by over-expressing the tlr4 gene alone using gene amplification in transgenic mice. TLR4 increased expression, without any exogenous insult and induced a similar lupus-like autoimmune disease (Liu B., et al 2006 J. of Immunol 177:6880-6888). The dependence of TLRs' activity on gp96 function may explain these corresponding results which suggest that chronic stimulation by gp96 or by TLRs may contribute to the development of autoimmune disorders. Indeed, data originating predominantly from animal models of autoimmune disease and circumstantial data from human patients suggest that inappropriate activation of TLR pathways by endogenous or exogenous ligands may lead to the initiation and/or maintenance of autoimmune responses and tissue injury (Papadimitraki, E. V. et al. 2007 J. of autoimmunity 29:310-318). Furthermore, agents that are currently used to treat autoimmune diseases, such as chloroquine and hydroxychloroquine, were demonstrated to block TLR signaling, which may explain their efficacy.
The involvement of TLRs in the pathogenesis of autoimmune disorders prompted their development as promising targets for therapeutic agents. Specific TLR antagonists are in preclinical and clinical development as therapeutics for various inflammatory disorders and autoimmune diseases (Gearing A. J. H, 2007 Immunology and Cell Biol. 85:490-494; Tse K & Horner A. 2007 Ann Rheum Dis. 66(Suppl III):iii77-80). Inhibition of gp96 activity may also be used as a therapeutic target to lessen TLRs malfunctioning in various disease states. Specific examples of specific autoimmune diseases or animal models that have been targeted by inhibitors of TLRs as promising therapeutic agents include: Systemic Lupus Erythematosus (SLE)—a dual inhibitor of TLR7 and TLR9 prevented the progression of a lupus-like disease when injected to lupus prone mice. Inflammatory bowel diseases (IBD) and other chronic gastrointestinal inflammation conditions where TLR4 plays a role—an antagonist of TLR4 inhibited the development of moderate-to-severe disease in two mouse models of colonic inflammation (Fort et al, 2005 J. Immunology 174:6416-6423). TLR4 might also serve as a target in the treatment of rheumatoid arthritis as inhibition of TLR4 suppressed the severity of experimental arthritis (Abdollahi-Roodsaz et al, 2007 Arthritis & Rheumatism 56:2957-2967).
TLRs are present on a number of cell types believed to be involved in the development of allergic sensitization and the early asthmatic reaction. Indeed, experimental studies have largely demonstrated the implication of TLRs in both development and control of the allergic reaction. These results have demonstrated the clinical potential of pharmacologic interventions that target TLRs for the prevention and treatment of allergic diseases (Bauer S. et al. 2007 Immunobiology 212:521-33).
TLRs activation also contributes to the development and progression of atherosclerosis, cardiac dysfunction in sepsis, congestive heart failure and ischemic injury. The involvement of TLRs in these conditions indicates that TLR inhibition could have protective effects in cardiovascular diseases as well as systemic and intragraft inflammatory responses that occur after cold ischemia-reperfusion in the setting of organ transplantation (Frantz S. et al. 2007 Nature Clinical Practice 4:444-454). Furthermore, high correlation was found between high TLR2 and TLR4 expression on circulating monocytes and liver transplantation recipients with acute rejection compared with those in clinically stable, normal liver function. These results suggested that activation of innate immunity in liver transplant recipients through TLR2 and TLR4 contributes to the development of acute allograft rejection after liver transplantation (Deng J. F. et al. 2007 Transplant Proc. 39:3222-3224).
Toll-like receptor antagonists, together with antibiotics, may delay or prevent infection-associated preterm birth. Pretreatment with TLR4 antagonist inhibited LPS-induced preterm uterine contractility, cytokines, and prostaglandins in rhesus monkeys (Waldorf K. M. et al. 2008 Reprod Sci. 15:121-127).
Recent studies show that TLRs are also expressed on a wide variety of tumors suggesting that TLRs may play important role in tumor progression. Activation of tumor cell TLRs not only promotes tumor cell proliferation and resistance to apoptosis, but also enhances tumor cell invasion and metastasis by regulating metalloproteinases and integrins. Moreover, the activation of TLR signaling in tumor cells induces the synthesis of proinflammatory factors and immunosuppressive molecules, which enhance the resistance of tumor cells to cytotoxic lymphocyte attack, leading to tumor evasion from immune surveillance. Thus, the neoplastic process seems to exploit TLR signaling pathways to advance cancer progression as well as immune evasion, suggesting that targeting tumor TLR signaling pathways may open novel therapeutic avenues (Huang B. et al., 2008 Oncogene 27:218-224).
Sepsis and septic shock, its more severe form, have shown alarming increases in incidence and a persistently high mortality rate, despite technological advancement allowing adequate support of vital functions in intensive care units. There is increasing evidence that TLRs play a key role in the mediation of systemic responses to invading pathogens during sepsis. Blockade of TLRs signaling suggests new potential therapeutic strategies for treating sepsis (Tsujimoto H. et al. 2008 Shock 29:315-321). Furthermore, HSPs, including gp96, can bind LPS directly, and are involved in the amplification of the immune response to endotoxin which takes place during sepsis (Triantafilou and Triantafilou 2004 Biochem. Soc. Trans. 32:636-639; Reed et al 2003 J. Biol. Chem. 278: 31853-31860).
To date, there are no known therapeutic agents which inhibit gp96. However, as demonstrated above, most investigative strategies are currently aimed at developing TLRs antagonists capable of inhibiting innate immune responses for the potential treatment of a vast array of immuno-regulated disorders. Another strategy is targeting CD91, the receptor for gp96. Small molecule inhibitors of CD91 or HSPs binding fragments of CD91 are being developed for the potential treatment of autoimmune disorders like multiple sclerosis, SLE and insulin dependent diabetes. Based on its critical role in the expression and function of various TLRs, antagonizing gp96 may be a more effective approach for the treatment of these disease conditions.